Increased oxidative stress in skeletal muscle of HFHSD mice

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Since mitochondrial alterations were observed in HFHSD mice only when they were hyperglycaemic and hyperlipidaemic, and since both glucose and lipids are known to induce oxidative stress, we tested whether ROS levels were increased during HFHSD feeding. Plasma H₂O₂ concentrations (Table 1) and muscular protein carbonylation levels (Figure 4A), a marker of protein oxidation, were elevated in 16 week HFHSD mice compared to 16 week SD mice. No difference was observed after 4 weeks of diet (Figure 4A).

            In addition, mRNA levels of uncoupling proteins 2 and 3 (markers reflecting increased mitochondrial ROS production in condition of lipid oversupply (17)) and of four subunits of NAD(P)H oxidase (gp91, p67, p40, p47) were induced in skeletal muscle of 16 weeks HFHSD mice, suggesting an increase of both mitochondrial and cytoplasmic ROS production (Figure 4B). Only the p67 subunit of NAD(P)H oxidase was significantly induced after 4 weeks of HFHSD feeding. Concerning the antioxidant system, the mRNA levels of glutathione reductase and catalase were increased in skeletal muscle of 16 week HFHSD mice, whereas expression of other antioxidant enzymes, including glutathione peroxidase, superoxide dismutase 2, peroxiredoxin 3 and peroxiredoxin 5 were not modified (Figure 4B). None of these genes showed a modification of expression after 4 weeks of HFHSD.

            Exposure to ROS leads to apoptosis and cell damages in a variety of experimental systems. As shown in Figure 4C, we observed an increase in cytochrome C levels in the cytosol with a concomitant decrease in the mitochondria fraction in skeletal muscle of 4 week HFHSD mice, indicating a cytochrome C release from mitochondria. Due to the strong alterations in the number and functions of mitochondria in the muscle of 16 weeks HFHSD-fed mice, this phenomenon was difficult to evidence after 16 weeks of feeding (Figure 4C). Nevertheless, the activity of caspase 3, another index of apoptosis, was markedly increased in skeletal muscle of 16 week HFHSD mice (90%, p<0.05), whereas no difference was observed after 4 weeks of diet (Figure 4D).           

We have also investigated ROS production and mitochondrial dysfunction in KKA^y mice, a genetic model of obesity and diabetes. KKA^y mice were obese, hyperglycaemic, hyperinsulinaemic, and hypertrygliceridaemic, compared to age-matched control mice (Table S1), but they showed normal plasma FFA levels (Table S1).  Plasma H₂O₂ levels were increased in KKA^y mice compared to C57Bl/6 mice (Table S1), but not skeletal muscle protein carbonylation (Figure S4A). Interestingly, in contrast to the HFHSD model, mitochondrial density and structure were not altered in KKA^y mice compared to age-matched control mice (Figures S4B and S4C). Taken together, these data suggest that oxidative stress in skeletal muscle is a determinant parameter for mitochondrial alterations in diabetic mice.